Ex vivo B-cell cultures are important tools in current biological and medical applications. One important application is culturing antibody-producing cells in order to harvest antibodies, preferably monoclonal antibodies. Monoclonal antibodies (mAbs) represent multiple identical copies of a single antibody molecule. Amongst the benefits of mAbs is their specificity for the same epitope on an antigen. This specificity confers certain clinical advantages on mAbs over more conventional treatments while offering patients an effective, well-tolerated therapy option with generally low side effects. Moreover, mAbs are useful for biological and medical research.
Mature B-cells can be cultured in vitro under conditions that mimic some key aspects of the germinal center (GC) reaction; that is, activation of B-cells with CD40 ligand (L) and the presence of cytokines like interleukin (IL)-4, IL-10 or IL-21. While B-cells cultured with CD40L, IL-2 and IL-4 produce very little Ig, addition of IL-21 leads to differentiation to plasma cells accompanied by high Ig secretion. Although this in vitro system has proven useful to study some aspects of B-cell differentiation, both naïve IgD+ B-cells and switched IgD-memory B-cells eventually differentiate into terminally differentiated plasma cells, which is accompanied by cell cycle arrest precluding the generation of long-term antigen-specific BCR-positive cell lines.
Recent advances have provided insight into how multiple transcription factors, including B-lymphocyte-induced maturation protein 1 (BLIMP1) and B-cell lymphoma (BCL)6 control development of GC B-cells into terminally arrested, antibody-producing plasma cells. The transcriptional repressor BCL6 has been shown to prevent plasma cell differentiation. BCL6 is highly expressed in GC B-cells where it facilitates expansion of B-cells by down-regulating p53 and prevents premature differentiation of GC cells into plasma cells by negatively regulating BLIMP1.
An improved method for generating an antibody-producing plasmablast-like B-cell was recently described in WO 2007/067046, which is hereby incorporated by reference. According to this method, the amount of BCL6 and a Bcl-2 family member, preferably Bcl-xL, are modulated in a B-cell, preferably a memory B-cell, to generate an antibody-producing plasmablast-like B-cell. In WO 2007/067046, the amount of BCL6 and/or Bcl-xL expression product is either directly or indirectly influenced. Preferably, the amounts of both BCL6 and Bcl-xL expression products within the antibody-producing cell are increased, since both expression products are involved in the stability of an antibody-producing B-cell. Bcl-xL is a member of the anti-apoptotic Bcl-2 family. Processes that are controlled by the Bcl-2 family, which includes both pro- and anti-apoptotic proteins, relate to the mitochondrial pathway of apoptosis. This pathway proceeds when molecules sequestered between the outer and inner mitochondrial membranes are released into the cytosol by mitochondrial outer membrane permeabilization. The pro-apoptotic family members can be divided in two classes. The effector molecules Bax and Bak, which contain so-called Bcl-2 homology domain 3 (BH3) domains, are involved in permeablilizing the outer mitochondrial membrane by forming proteolipid pores; the pro-apoptotic BH3-only proteins (Bad, Bik, Bim, Bid, Hrk, Bmf, bNIP3, Puma and Noxa) function upon different cellular stresses by protein-protein interactions with other (anti-apoptotic) Bcl-2 family members.
Anti-apoptotic Bcl-2 family members Bcl-2, Bcl-xL, Bcl-w, A1 and Mcl-1 are generally integrated with the outer mitochondrial membrane. They directly bind and inhibit the pro-apoptotic Bcl-2 proteins to protect mitochondrial membrane integrity.
In such a method, it is further preferred that the antibody-producing plasmablast-like B-cell is incubated with IL 21 and CD40L. A B-cell, such as an antibody-producing plasmablast-like B-cell, is preferably cultured in the presence of CD40L since replication of most B-cells is favored by CD40L. It is furthermore preferred that STAT3 is activated in the antibody-producing B-cell. Activation of STAT3 can be achieved in a variety of ways. Preferably, STAT3 is activated by providing an antibody-producing cell with a cytokine. Cytokines, being naturally involved in B-cell differentiation, are very effective in regulating STAT proteins. Very effective activators of STAT3 are IL-2, IL-10, IL-21 and IL-6, but also IL-7, IL-9, IL-15, IL-23 and IL-27 are known to activate STAT3. Additionally, or alternatively, STAT3 activation is accomplished by transfer into a B-cell of a nucleic acid encoding a mutant of STAT3 that confers constitutive activation to STAT3. (Sean A. Diehl, Heike Schmidlin, Maho Nagasawa, Simon D. van Haren, Mark J. Kwakkenbos, Etsuko Yasuda, Tim Beaumont, Ferenc A. Scheeren, Hergen Spits STAT3-mediated up-regulation of BLIMP1 is coordinated with BCL6 down-regulation to control human plasma cell differentiation. J. Immunol. 2008 vol. 180 (7) pp. 4805-15.)
Most preferably, IL-21 is used, since IL-21 is particularly suitable for influencing the stability of an antibody-producing plasmablast-like B-cell. In addition to up-regulating STAT3, IL-21 is capable of up-regulating BLIMP1 expression even when BLIMP1 expression is counteracted by BCL6. With the methods disclosed in WO 2007/067046, it has become possible to increase the replicative life span of an antibody-producing cell since it is possible to maintain a B-cell in a developmental stage wherein replication occurs. In earlier ex vivo B-cell cultures, the replicative life span was only a few weeks to two months. During this time, the cultured cells lose their capability of replicating and die. With a method as disclosed in WO 2007/067046, however, it has become possible to prolong the replicative life span of antibody-producing memory B-cells, so that ex vivo cultures are generated comprising plasmablast-like B-cells that are capable of replicating and producing antibody.
Although these methods enable the production of antibodies that efficiently target an antigen of interest, improvement of antibody characteristics, such as binding affinity, is often desired. Binding characteristics are, therefore, regularly altered by introducing mutations in the encoding nucleic acid, preferably in the CDR encoding region, and testing the resulting antibodies. This is, however, time consuming. Alternative methods for obtaining high-affinity antibodies are, therefore, desired.